Data processing of RNAseq data

Overview of sample characteristics:

## 377 samples selected for analysis
## 428 files found
## The following files do not have a matching metadata entry:
##  [1] "X500006_2"       "X500016_2"       "X500017_2"       "X500082_2"       "X500110_2"       "X500126_2"       "X500161_2"       "X500224_2"       "X500471_2"       "X500500_2"       "X500519_2"       "X500523_2"       "X500531_2"       "X500533_2"       "X500534_2"       "X500535_2"       "X510008_2"       "X510010_2"       "X533070_2"       "X533071_2"       "X533181_2"       "X533182_2"       "X541038_2"       "X541183_2"       "X541728_2"       "X541980_2"       "X550004_2"       "X590013_2"       "X590015_2"       "X590048_2"       "X590098_2"       "X590113_2"       "X500133_2"       "X500544_2"       "X500545_2"       "X500576_2"       "X500584_2"       "X500586_2"       "X500587_2"       "X500588_2"       "X500589_2"       "X500590_2"       "X510033_2"       "X541051_2"       "X541052_2"       "X541091_2"       "X541191_2"       "X598101_2"       "S00727_V07059_2" "S01248_V07399_2" "AA04447"
## All expected files found.

Sample QC filtering

Samples with a PC1 > 2 (the red lines) and/or PC2 zscore > 2.5 (the green lines) were dropped from analysis. Points in red represent samples that were dropped.

Removing the following samples:
sample_name visit_ref subject_ref sex ethnicity age
AA04628 BLAST|0001|2015-02-02 BLAST|0001 Female [EA] 33
AA04629 BLAST|0001|2015-02-12 BLAST|0001 Female [EA] 33
AA04630 BLAST|0001|2015-03-17 BLAST|0001 Female [EA] 33
AA04633 BLAST|0001|2015-05-22 BLAST|0001 Female [EA] 33
AA04634 BLAST|0001|2015-06-04 BLAST|0001 Female [EA] 33
AA04636 BLAST|0001|2015-07-09 BLAST|0001 Female [EA] 33
AA04637 BLAST|0001|2015-08-04 BLAST|0001 Female [EA] 33
AA04759 BLAST|0014|2015-10-08 BLAST|0014 Male [EA] 37
AA04760 BLAST|0016|2015-11-19 BLAST|0016 Female [EA][H] 51
AA04762 BLAST|0016|2016-01-14 BLAST|0016 Female [EA][H] 52
AA04763 BLAST|0017|2016-01-19 BLAST|0017 Female [EA] 49
AA04773 BLAST|0017|2016-08-16 BLAST|0017 Female [EA] 49
AA04782 BLAST|0018|2016-06-14 BLAST|0018 Female [EA] 56
AA04841 BLAST|0027|2018-06-07 BLAST|0027 Female [EA] 37
AA04873 BLAST|0030|2018-07-30 BLAST|0030 Female [AI] 43
AA04874 BLAST|0030|2018-08-09 BLAST|0030 Female [AI] 43
AA04879 BLAST|0030|2019-04-10 BLAST|0030 Female [AI] 44
AA04894 BLAST|0031|2019-05-23 BLAST|0031 Female [EA] 34
X541052 ORDRCC|541052|2019-01-24 ORDRCC|541052 Female [EA] 66
X541980 ORDRCC|541980|2015-01-27 ORDRCC|541980 Female [AA] 45
AA02818 MDFL|541038|2017-04-20 MDFL|541038 Female [EA] 32
AA02821 MDFL|500531|2017-06-27 MDFL|500531 Female [AA] 60
AA02817 MDFL|500519|2017-04-20 MDFL|500519 Female [EA] 33
AA02819 MDFL|500006|2017-05-01 MDFL|500006 Female [AA] 42

Overall correlation between samples

Data quality

MA-plot

This MA plot is of shrunken log2 fold changes (using the apeglm package), in which actual fold changes are adjusted to compensate for the high variablity of genes with few counts.

Dots in red indicate a significant expression difference between Control and SLE.

Dispersion estimates

Surrogate Variable Analysis

Surrogate variables are unknown, unmodeled, or latent sources of noise hidden in the data. The sva package was used to estimate the first three surrogate variables which were then introduced into the DESeq design formula to control for their effects.

K-means clustering optimization

To determine the optimal k, the gap statistic was calculated for k = (3,20) (k = 2 was ignored because it just returns the two disease clusters). A higher coefficient means better intracluster simliarity and intercluster differences.

Sample Sanity check

To determine if there is a gross mixup of samples, examine the expression of sex-specific transcripts to ensure that only female-subject samples express “XIST” and male-subject samples "DDX3Y:

There appears to be one sample that is lacking a label but appears to express high levels of the female markers XIST and ZFX as well as the male markers PCDH11Y and PRORY. There’s also a couple of other samples that may have a mix of male and female markers, but otherwise no signs of gross sample mixup.

## ─ Session info ───────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────
##  setting  value                       
##  version  R version 4.0.4 (2021-02-15)
##  os       Ubuntu 20.04 LTS            
##  system   x86_64, linux-gnu           
##  ui       X11                         
##  language (EN)                        
##  collate  en_US.UTF-8                 
##  ctype    en_US.UTF-8                 
##  tz       Etc/UTC                     
##  date     2021-03-22                  
## 
## ─ Packages ───────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────
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##  pROC                   1.17.0.1   2021-01-13 [1] RSPM (R 4.0.3)                           
##  prodlim                2019.11.13 2019-11-17 [1] RSPM (R 4.0.3)                           
##  progress               1.2.2      2019-05-16 [1] RSPM (R 4.0.4)                           
##  proxy                  0.4-25     2021-03-05 [1] RSPM (R 4.0.3)                           
##  purrr                * 0.3.4      2020-04-17 [1] RSPM (R 4.0.3)                           
##  qvalue                 2.22.0     2020-10-27 [1] RSPM (R 4.0.4)                           
##  R6                     2.5.0      2020-10-28 [1] RSPM (R 4.0.3)                           
##  randomForest         * 4.6-14     2018-03-25 [1] RSPM (R 4.0.3)                           
##  RColorBrewer         * 1.1-2      2014-12-07 [1] RSPM (R 4.0.3)                           
##  Rcpp                   1.0.6      2021-01-15 [1] RSPM (R 4.0.4)                           
##  RcppParallel           5.0.3      2021-02-24 [1] RSPM (R 4.0.4)                           
##  RCurl                  1.98-1.2   2020-04-18 [1] RSPM (R 4.0.4)                           
##  readr                * 1.4.0      2020-10-05 [1] RSPM (R 4.0.4)                           
##  readxl               * 1.3.1      2019-03-13 [1] RSPM (R 4.0.3)                           
##  recipes              * 0.1.15     2020-11-11 [1] RSPM (R 4.0.3)                           
##  registry               0.5-1      2019-03-05 [1] RSPM (R 4.0.4)                           
##  rematch2               2.1.2      2020-05-01 [1] RSPM (R 4.0.3)                           
##  reprex                 1.0.0      2021-01-27 [1] RSPM (R 4.0.4)                           
##  reshape2               1.4.4      2020-04-09 [1] RSPM (R 4.0.4)                           
##  reticulate             1.18       2020-10-25 [1] RSPM (R 4.0.4)                           
##  rio                    0.5.26     2021-03-01 [1] RSPM (R 4.0.4)                           
##  rlang                * 0.4.10     2020-12-30 [1] RSPM (R 4.0.3)                           
##  rmarkdown            * 2.7        2021-02-19 [1] RSPM (R 4.0.4)                           
##  rngtools               1.5        2020-01-23 [1] RSPM (R 4.0.4)                           
##  rpart                  4.1-15     2019-04-12 [2] CRAN (R 4.0.4)                           
##  rprojroot              2.0.2      2020-11-15 [1] RSPM (R 4.0.3)                           
##  rsample              * 0.0.9      2021-02-17 [1] RSPM (R 4.0.3)                           
##  RSpectra               0.16-0     2019-12-01 [1] RSPM (R 4.0.4)                           
##  RSQLite                2.2.4      2021-03-12 [1] RSPM (R 4.0.3)                           
##  rstatix              * 0.7.0      2021-02-13 [1] RSPM (R 4.0.4)                           
##  rstudioapi             0.13       2020-11-12 [1] RSPM (R 4.0.3)                           
##  rsvd                   1.0.3      2020-02-17 [1] RSPM (R 4.0.4)                           
##  rvcheck                0.1.8      2020-03-01 [1] RSPM (R 4.0.4)                           
##  rvest                  1.0.0      2021-03-09 [1] RSPM (R 4.0.4)                           
##  S4Vectors            * 0.28.1     2020-12-09 [1] RSPM (R 4.0.4)                           
##  sass                   0.3.1      2021-01-24 [1] RSPM (R 4.0.4)                           
##  scales               * 1.1.1      2020-05-11 [1] RSPM (R 4.0.3)                           
##  scatterpie             0.1.5      2020-09-09 [1] RSPM (R 4.0.4)                           
##  sessioninfo            1.1.1      2018-11-05 [1] RSPM (R 4.0.3)                           
##  shadowtext             0.0.7      2019-11-06 [1] RSPM (R 4.0.4)                           
##  snakecase              0.11.0     2019-05-25 [1] RSPM (R 4.0.4)                           
##  snow                   0.4-3      2018-09-14 [1] RSPM (R 4.0.4)                           
##  storr                  1.2.5      2020-12-01 [1] RSPM (R 4.0.4)                           
##  stringi                1.5.3      2020-09-09 [1] RSPM (R 4.0.3)                           
##  stringr              * 1.4.0      2019-02-10 [1] RSPM (R 4.0.3)                           
##  SummarizedExperiment * 1.20.0     2020-10-27 [1] RSPM (R 4.0.4)                           
##  survival               3.2-7      2020-09-28 [2] CRAN (R 4.0.4)                           
##  sva                  * 3.38.0     2020-10-27 [1] Bioconductor                             
##  svglite                2.0.0      2021-02-20 [1] RSPM (R 4.0.4)                           
##  systemfonts            1.0.1      2021-02-09 [1] RSPM (R 4.0.4)                           
##  tibble               * 3.1.0      2021-02-25 [1] RSPM (R 4.0.3)                           
##  tidygraph              1.2.0      2020-05-12 [1] RSPM (R 4.0.4)                           
##  tidymodels           * 0.1.2      2020-11-22 [1] RSPM (R 4.0.3)                           
##  tidyr                * 1.1.3      2021-03-03 [1] RSPM (R 4.0.4)                           
##  tidyselect             1.1.0      2020-05-11 [1] RSPM (R 4.0.4)                           
##  tidyverse            * 1.3.0      2019-11-21 [1] RSPM (R 4.0.4)                           
##  timeDate               3043.102   2018-02-21 [1] RSPM (R 4.0.3)                           
##  tune                 * 0.1.3      2021-02-28 [1] RSPM (R 4.0.3)                           
##  tweenr                 1.0.1      2018-12-14 [1] RSPM (R 4.0.4)                           
##  tximport             * 1.18.0     2020-10-27 [1] RSPM (R 4.0.4)                           
##  txtq                   0.2.3      2020-06-23 [1] RSPM (R 4.0.4)                           
##  utf8                   1.2.1      2021-03-12 [1] RSPM (R 4.0.3)                           
##  uuid                   0.1-4      2020-02-26 [1] RSPM (R 4.0.3)                           
##  uwot                 * 0.1.10     2020-12-15 [1] RSPM (R 4.0.4)                           
##  vctrs                  0.3.6      2020-12-17 [1] RSPM (R 4.0.3)                           
##  viridis              * 0.5.1      2018-03-29 [1] RSPM (R 4.0.3)                           
##  viridisLite          * 0.3.0      2018-02-01 [1] RSPM (R 4.0.4)                           
##  webshot                0.5.2      2019-11-22 [1] RSPM (R 4.0.4)                           
##  WGCNA                * 1.70-3     2021-02-28 [1] RSPM (R 4.0.4)                           
##  withr                  2.4.1      2021-01-26 [1] RSPM (R 4.0.3)                           
##  workflows            * 0.2.2      2021-03-10 [1] RSPM (R 4.0.3)                           
##  xfun                   0.22       2021-03-11 [1] RSPM (R 4.0.3)                           
##  XML                    3.99-0.5   2020-07-23 [1] RSPM (R 4.0.4)                           
##  xml2                   1.3.2      2020-04-23 [1] RSPM (R 4.0.3)                           
##  xtable                 1.8-4      2019-04-21 [1] RSPM (R 4.0.4)                           
##  XVector                0.30.0     2020-10-27 [1] RSPM (R 4.0.4)                           
##  yaml                   2.2.1      2020-02-01 [1] RSPM (R 4.0.3)                           
##  yardstick            * 0.0.7      2020-07-13 [1] RSPM (R 4.0.3)                           
##  zip                    2.1.1      2020-08-27 [1] RSPM (R 4.0.3)                           
##  zlibbioc               1.36.0     2020-10-27 [1] RSPM (R 4.0.4)                           
## 
## [1] /usr/local/lib/R/site-library
## [2] /usr/local/lib/R/library